Method for the purification and aqueous fiber spinning of spider silks and other structural proteins

ABSTRACT

Methods are described for the purification and spinning of recombinant and non-recombinant proteins. Specifically, the lysis of bacteria and purification of silk proteins occur in a single solution of organic acid. Bacterial proteins are hydrolyzed while the silk protein remains intact. Silk proteins remain soluble as they are concentrated into a aqueous-based mixture for fiber spinning.

STATEMENT OF GOVERNMENTAL INTEREST

The present invention may be used or licensed by the United StatesGovernment for Governmental purposes without the payment of any royalty.

FIELD OF THE INVENTION

The present invention relates to methods for purifying and spinningspider silks and other structural proteins. Specifically, organic acidsare used to lyse recombinant cells or other biological samples (such asnon-recombinantly derived cells), and significantly enrich the purityand yields of structural proteins by hydrolyzing many of themacromolecules, while leaving the structural proteins intact. In thecase of silk proteins, the resulting lysate is further purified byion-exchange or affinity chromatography and processed into anaqueous-based mixture for fiber spinning.

BACKGROUND

Spiders produce a number of silks for different functions and aretherefore useful organisms to produce a variety of structural proteins.The structural fibers of the golden orb-weaver spider (Nephilaclavipes), are extremely strong and flexible, and are able to absorbimpact energy from flying insects without breaking. Dragline silk fibersdissipate energy over a broad area and balance stiffness, strength andextensibility. In addition, silk proteins have very low antigenicity.Therefore, silk fibers are well suited for light weight, highperformance fiber, composite and medical applications. The compositionof these proteins is mainly glycine, alanine, and other short side chainamino acids, which form anti-parallel beta-pleated sheets by hydrogenbonding and hydrophobic interactions; Lucas et al., Discovery 25:191964. Many spider silks are resistant to digestion by proteolyticenzymes; Tillinghast and Kavanaugh, Journal of Zoology 202:212 1977, andinsoluble in dilute acids and bases; Mello et al., American ChemicalSociety Symposium Series 544, Silk Polymers: Materials, Science andBiotechnology pp 67-79, 1995. Spiders are not capable of producingsufficient quantities of proteins to enable a practical use of theirpotential. To solve this problem, recombinant spider silks have beenexpressed in E.coli; Arcidiacono et al., Applied Microbiology andBiotechnology 49:31 1998; Fahnestock and Irwin, Applied Microbiology andBiotechnology 47:23, 1997; Fahnestock and Irwin, Applied Microbiologyand Biotechnology 47:33 1997; Lewis et al., Protein Expression andPurification 7:400, 1996; Prince et al., Biochemistry 34:10879 1995.However, the purification and preparation of a protein for fiberspinning has been particularly difficult due to the solubilitycharacteristics and unique properties of spider silk and otherstructural proteins.

Native Nephila clavipes spider dragline fiber has been partiallysolubilized in hexafluoroisopropanol (HFIP) and dried to a film. A 2.5%(w/w) solution of the film in HFIP was used for spinning; Jelinski etal., Macromolecules 31:6733 1998. The spinning was conducted with asyringe pump at 6 uL/s by forcing the HFIP solution through thespinneret into a coagulation bath.

Affinity chromatography has been used for purification by binding to anengineered tag in the recombinant protein while washing away bacterialproteins; Arcidiacono et al., Applied Microbiology and Biotechnology49:31 1998; Fahnestock and Irwin, Applied Microbiology and Biotechnology47:23 1997; Lewis et al., Protein Expression and Purification 7:4001996; Prince et al., Biochemistry 34:10879 1995. One commonly used tagis a hexa-histidine tag, that binds with high affinity to a nickelaffinity resin. After washing away the bacterial proteins, the taggedrecombinant protein can be eluted from the resin. There are severaldisadvantages to this method: 1) large volumes of denaturing buffers arerequired, involving multiple steps and time; 2) not all target proteinis recovered; 3) other bacterial proteins remain, often requiringadditional purification (i.e., high-performance liquid chromatography(HPLC)); 4) the method is not easily scaled-up; 5) and the presence ofan affinity tag on the recombinant protein may increase its antigenicityand interfere with the necessary molecular alignment required for highstrength fibers. Accordingly, there is a continuing need to develop newmethods for the purification of structural proteins, spinning of silkfibers lacking the engineered tag and enabling the assembly ofmacromolecular structures without potential interferences.

SUMMARY OF THE INVENTION

As a solution to the above-related deficiencies in the prior art, thepresent invention contemplates using organic acids to purify recombinantspider silks or other non-recombinant structural proteins from E. colibacteria while removing the unwanted bacterial proteins. The inventionis based on the unique solubilization and stability characteristics ofthese proteins, which are resistant to acid hydrolysis for prolongedperiods of time at room temperature, while many globular proteins arenot. Purified protein solutions can be processed into a spinnableaqueous-based mixture for the production of fibers. The presentinvention also contemplates an aqueous protein spinning method thatclosely mimics the natural spinning process of the spider and has thepotential to produce fibers with properties that may resemble or improveupon those of natural silk fibers. The present invention represents thefirst known example of an aqueous process for the spinning of silkproteins into fibers. Furthermore, this invention is the only knownreport, to date, of spinning recombinant silk proteins into fibers. Thepresent invention displays numerous advantages over the background art,including a purification method with organic acids containing fewersteps, requiring less time and smaller volumes of reagents. The presentinvention also results in better recovery of protein at a higher purity.For example, the (SP1)₇ protein can be recovered at a level of 150 mg/L,compared to the 7mg/L recovery rate by the current art (see Prince etal., supra). While not limited to any mechanism by which a recovery isachieved, it is believed that lower protein recovery rates by thetraditional methods are caused, in part, from incomplete binding of theprotein to the affinity resin. Such traditional techniques include, butare not limited to, ion exchange chromatography and affinitychromatography. The inability of these proteins to bind to the resin ismost likely due to a high degree of secondary structure even in thepresence of high concentrations of denaturant. Sample purity from thepresent invention has been obtained in the range of 94-97% as determinedby amino acid analysis (see Examples 1 and 2, infra). The current artresults in a wide and inconsistent range of purity ranging from 70%(Prince et al., supra), to 99% (Lewis et al., 1996, supra). While highsample purity is possible using current art by affinity chromatography,the presence of the histidine affinity tag significantly increases theantigenicity of the protein and adversely affects the properties offibers, films, or other materials by disrupting the proper molecularorientation required within the material. Also, in many cases thecurrent art results in samples still contaminated by other bacterialproteins, requiring additional purification such as HPLC (Prince et al.;Lewis et al., supra). Finally, the methods of the present invention areeasily scaled-up, and fibers are spun in an environmentally benignsolution reducing hazardous waste accumulation and cost. For example,the present invention contemplates the spinning of silk proteins in anenvironmentally innocuous aqueous based system. In one embodiment of thepresent invention, a solution of an organic acid is used to effect thelysis of bacteria and initiate purification of recombinant silks andnative structural proteins. Globular proteins are hydrolyzed while thesilk protein remains intact. Silk proteins remain and are concentratedinto an aqueous-based mixture for fiber spinning. The embodiment maycomprise the following steps: a) resuspension of the cell pellet inconcentrated organic acid and dilution to 2.3N in water (+/−denaturantand/or surfactant) to form a homogeneous mixture; b) incubation at roomtemperature 1 hour with stirring and centrifugation to remove celldebris; c) reduction of volume, 10-100 fold by ultrafiltration andremoval of insoluble material by centrifugation; d) dialysis and removalof insoluble material by centrifugation; e) purification by ion exchangechromatography and dialysis into processing buffer; f) concentration ofsolution to 11-40% (w/w) protein by ultrafiltration and spinningsolution into fibers. While this embodiment is given for guidance, thoseof skill in the art may choose to add or delete certain steps whileremaining within the spirit and scope of the present invention. Forexample, the purification methodology may be employed with or withoutthe spinning of the fiber solution. Several native and recombinantstructural proteins have been purified by this method. Any biologicalsample containing a structural protein of interest, native orrecombinant, is amenable to the methodology outlined in the invention.Examples of biological samples may include, but are not limited to, E.coli cells, other bacterial cells, eukaryotic cells, a medium in which astructural protein has been secreted, bone, tissues or organs. And whilemany variables have been examined and optimized throughout the process,each variable and optimization exemplify variations of the overallgeneral method. Choosing among the various parameters is highlydependent on the protein being prepared. Table 1 below lends guidance tothose of skill in the art.

TABLE 1 Variables Explored Conclusion Lysis 1. Type of acid Protein:(4 + 1)₄ Acid: Propionic Protein: (Spl)₇ Acid: Formic Protein: NcDSAcid: Formic Protein: OmpF Acid: Valeric Protein: Recognin Acid: Valeric2. Volume acid/g cells Increased acid volume generally decreases (2-100ml/g) purity 3. Acid strength Full strength is best (23N Formic, 13N(0.5-23N) Propionic) for lysis. 4. Length of lysis (30 min- 1 hr ispreferred overnight) 5. Temperature of lysis No effect (25° C.-37° C.)6. Lysis under denaturing Solubility improves, purity decreasesconditions 7. Lysis in the presence of No effect on purity detergentsPurification 1. Lysate purification by Chromatography successfullypurifies chromatography target (affinity, ion exchange). Processing 1.Urea concentration in the 1M urea improves solubility slightlyprocessing buffer (160 mM vs 1M) 2. Ionic strength of the IncreasingNaCl concentration causes processing buffer precipitation (20-100 mMNaCl) Spinning 1. Spin aqueous-based Protein dependent mixture proteinconcentration (11-35%) 2. Age of aqueous-based Spinnability changes asthe aqueous-based mixture (0-5 days) mixture ages 3. Temperature duringaging Higher temperatures accelerate (4-30° C.) changes in the solutionbehavior (i.e. spinnability and solubility) 4. Coagulation bath (70-90%Methanol percentage affects speed of fiber methanol) formation, fiberbehavior

A variety of embodiments are contemplated. In one embodiment, thepresent invention contemplates a method, comprising: a) providing: i) abiological sample comprising one or more structural polypeptides; andii) an acid; b) treating said sample with said acid under conditionssuch that said one or more polypeptides is recovered in a solution. Avariety of structural peptides are contemplated, including but notlimited to polypeptides selected from SEQ ID NO.: 2, SEQ ID NO.: 4, SEQID NO.: 6, SEQ ID NO.: 8, SEQ ID NO.:9, and SEQ ID NO.: 11. the peptidesmay be recombinant or native polypeptides.

A variety of acids are contemplated. Organic acids are preferred. In oneembodiment, the present invention contemplates one or more organic acidsselected from formic, acetic, propionic, butyric, and valeric acids.

It is the goal to produce fibers. Therefore, in one embodiment, themethod further comprises the step of manipulating said solution underconditions such that insoluble fibers are produced. Indeed, the presentinvention specifically contemplates the fibers produced according to theabove-described process.

The present invention specifically contemplates methods whereinrecombinant structural proteins are manipulated. In one embodiment, thepresent invention contemplates a method, comprising: a) providing: i)host cells expressing one or more recombinant structural polypeptides,and ii) a solution comprising an organic acid; b) treating said hostcells with said solution to create a mixture; c) removing insolublematerial from said mixture; and d) recovering said one or morerecombinant polypeptides in a solution. Again, a variety of peptides arecontemplated. In one embodiment, one or more polypeptides are selectedfrom SEQ ID NO.: 2, SEQ ID NO.: 4, SEQ ID NO.: 6, SEQ ID NO.: 8, SEQ IDNO.:9, and SEQ ID NO.: 11. Again, a variety of acids are contemplated,including but not limited to organic acids selected from formic acid,acetic acid, propionic acid, butyric acid, and valeric acid.

To produce fibers, the method involves manipulation of said recoveredone or more recombinant polypeptides in said solution under conditionssuch that insoluble fibers are produced. The present inventionspecifically contemplates the fibers themselves produced according tothe above-described process.

A variety of host cells are contemplated for recombinant production.Thus, in one embodiment the present invention contemplates a method,comprising: a) providing: I) bacterial cells expressing one or morerecombinant structural polypeptides, and ii) a solution comprising anorganic acid selected from formic acid, acetic acid, propionic acid,butyric acid, and valeric acid; b) treating said bacterial cells withsaid solution to create a mixture; c) removing insoluble material fromsaid mixture; and d) recovering said one or more recombinantpolypeptides in a solution. As noted above, a variety of peptides arecontemplated, including but not limited to one or more polypeptides isselected from SEQ ID NO.: 2, SEQ ID NO.: 4, SEQ ID NO.: 6, SEQ ID NO.:8, and SEQ ID NO.: 11.

To produce fibers, said recovered one or more recombinant polypeptidesare manipulated under conditions such that insoluble fibers areproduced. In a preferred embodiment, said manipulating comprises: a)concentrating said recovered one or more recombinant silk polypeptidesto create a concentrated solution; and b) forcing said concentratedsolution through a spinneret. The present invention specificallycontemplated the fibers themselves which are produced according to thisprocess.

In sum, the present invention contemplates a method, which comprisesproviding a biological sample composed of a polypeptide and an acid, andmanipulating the biological sample under conditions such that thepolypeptide is substantially purified into an aqueous-based mixture.

The method, in several embodiments, includes using polypeptides that maybe selected from SEQ ID NO.: 2, SEQ ID NO.: 4, SEQ ID NO.: 6, SEQ IDNO.: 8, SEQ ID NO.: 9, and SEQ ID No.: 11 herein, although other aminoacid sequences are also contemplated.

In another embodiment of the present invention, the biological samplecomprises many types of polypeptides, including, but not limited to,recombinant and non-recombinant polypeptides. Structural polypeptides,such as silk polypeptides, are also contemplated.

In further embodiments of the present invention, organic acids are usedto manipulate aqueous-based mixtures under conditions such that themixtures may be processed into fibers. The organic acids that may beused include, but are not limited to, formic, acetic, propionic,butyric, and valeric acids. The present invention further contemplatesthe product that is achieved by the methods that are described herein.

While a variety of applications for the methods and products hereindescribed are contemplated, the applications are not limited. Forexample, the compositions of the present invention may comprise any typeof replacement for, or blended with, high strength light-weightsynthetic polymers (e.g., kevlar®) for applications such as manufactureof skis, skateboards, and tennis rackets. The method of the presentinvention can also be used to create a product that can be used as aprecursor to the construction of many materials, including, but notlimited to, films, fibers, woven articles (e.g., clothing), sutures,ballistic protection, parachutes and parachute cords.

DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

To facilitate an understanding of the invention, a number of Figures areincluded herein.

FIG. 1 presents the nucleic acid sequence of a recombinant silk protein(SEQ ID NO: 1) designated pQE(sp1)₇.

FIG. 2 presents a recombinant silk protein (SEQ ID NO: 2), designatedpQE(SP1)₇, that is the gene product of the nucleic acid sequencepresented in (SEQ ID NO: 1).

FIG. 3 presents the nucleic acid sequence of a recombinant silk protein(SEQ ID NO: 3) designated pQE[(SP1)₄/(SP2)₁]₄.

FIG. 4 presents a recombinant silk protein (SEQ ID NO: 4), designatedpQE[(SP1)₄/(SP2)₁]₄, that is the gene product of the nucleic acidsequence presented in (SEQ ID NO: 3).

FIG. 5 presents the nucleic acid sequence of a recombinant silk protein(SEQ ID NO: 5) designated pET[(SP1)₄/(SP2)₁]₄.

FIG. 6 presents a recombinant silk protein (SEQ ID NO: 6), designatedpET[(SP1)4/SP2)₁]₄ that is the gene product of the nucleic acid sequencepresented in (SEQ ID NO: 5).

FIG. 7 presents the nucleic acid sequence of a recombinant silk protein(SEQ ID NO: 7) designated pETNcDS.

FIG. 8 presents a recombinant silk protein (SEQ ID NO: 8), designatedpETNcDS, that is the gene product of the nucleic acid sequence presentedin (SEQ ID NO: 7).

FIG. 9 presents a bacterial membrane protein (SEQ ID NO: 9), designatedompF.

FIG. 10 presents the nucleic acid sequence of a recombinant structuralprotein. (SEQ ID NO: 10) designated Recognin B1.

FIG. 11 presents a recombinant structural protein (SEQ ID NO: 11),designated Recognin B1, that is the gene product of the nucleic acidsequence presented in (SEQ ID NO: 10).

FIG. 12 presents a polyacrylamide gel comparing acid lysis purificationof the recombinant silk protein pQE(Sp1)₇ to traditional denaturingmethod. The pQE(Sp1)₇ protein is enriched by acid lysis compared tolysis under denaturing conditions (e.g. 8M urea). Subsequent affinitychromatography purification by Ni-NTA of the formic acid lysate resultsin a yield comparable to the purification of the traditional denaturinglysate.

FIG. 13 represents a polyacrylamide gel depicting the QAE-Sephadexpurification scheme with a propionic acid extracted pET[(Sp1)₄/(Sp2)₁]₄protein sample.

FIG. 14 presents a polyacrylamide gel depicting the purification ofpET[(Sp1)₄/(Sp2)₁]₄ by lysis with propionic acid with 3M guanidine-HCland ion-exchange chromatography using QAE-Sephadex A50.

FIG. 15 represents a polyacrylamide gel depicting the purification ofompF, a native E. coli structural protein from a lyophilized E. colicell pellet. The cell pellet was extracted using valeric acid. Thisextraction procedure yielded a purity of approximately 85% based oncoomassie-blue staining.

FIG. 16 presents a polyacrylamide gel of Recognin B1, a recombinantcoiled coil structural protein. A cell pellet was lysed in either gelloading buffer, formic acid or valeric acid. Relative amounts of thecell pellet loaded onto the gel were 85, 400, 900 ug for the loadingbuffer, formic and valeric acid lysates, respectively. Acetic, propionicor butyric acids were unable to extract this protein.

FIG. 17 presents photomicrographs of a pETNcDS fiber spun from a proteinsolution of 25% (w/v) as determined by extinction coefficient. Fiberswere generated at a rate of at 10 ul/min in a 90% methanol coagulationbath. Consistent diameters of about 60 urn were observed. Underpolarizing light, the color changed uniformly from blue to yellow as theangle of light was changed indicating directional orientation in thefiber.

FIG. 18 presents photomicrographs of a fiber spun at a rate of 5 ul/mininto a 90% methanol coagulation bath from a 12.5% aqueous solution ofpQE[(Sp1)₄/(Sp2)₁]₄ viewed under a) white light and b) polarized lightwith a tint plate. The fibers present a consistent diameter of about 30um.

DEFINITIONS

To facilitate an understanding of the invention, a number of terms aredefined.

The term “aqueous”, as defined herein, refers to a water misciblesolution.

The term “aqueous-based mixture”, as defined herein, refers to a proteinin an aqueous solution. The mixture may be used for proteinpurification, fiber spinning, film formation or other materials.

The term “aqueous fiber spinning” refers to a process by which fibersare formed from an aqueous solution.

The terms “spin” “spinnable” as used herein, refers to a mixture that iscapable of forming a fiber and the fiber remains intact duringmanipulation (i.e. drawing and removal from a coagulation bath).

The term “biological sample”, as defined herein, refers to any samplecontaining a structural protein of interest, native or recombinant, thatis amenable to the methodology of the present invention. Examples ofbiological samples may include, but are not limited to, E. coli cells,other bacterial cells, eukaryotic cells, a medium where the structuralprotein has been secreted, bone, tissues or organs.

The term “recombinant protein”, as used herein, refers to the productproduced by expression of a recombinant DNA sequence in a foreign host.The (Sp1)7 protein, described herein in Example 1, exemplifies arecombinant protein.

The term “recombinant” or “recombining” refers to a nucleic acidsequence which is incorporated into a vector, e.g., into an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (e.g., a cDNA or agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other DNA sequences. This definition alsoincludes recombinant DNA which is part of a hybrid gene encodingadditional amino acid sequences.

The term “recombinant DNA vector” as used herein refers to DNA sequencescontaining a desired coding sequence and appropriate DNA sequencesnecessary for the expression of the operably linked coding sequence in aparticular host organism (e.g., bacteria). DNA sequences necessary forexpression in prokaryotes include a promoter, optionally an operatorsequence, a ribosome binding site and possibly other sequences.Eukaryotic cells are known to utilize promoters, polyadenylation signalsand enhancers.

The term “non-recombinant” refers to proteins that are derived by otherthan recombinant means. Non-recombinant protein may be structural ornon-structural. The E. coli OmpF membrane protein (described herein inExample 6), which is, in this case, a naturally occurring protein thatserves as an example of a non-recombinant protein.

The term “lyophilized pellet” represents a sample that is derived from abiological sample where the sample is frozen and dried under vacuum(−50° C. & 10-100 microns of Hg) to produce a powder.

The term “purified” or a “pure preparation” of a polypeptide, as usedherein, means a polypeptide that has been separated from other proteins,lipids, and nucleic acids with which it naturally occurs. Thepolypeptide is also separated from substances, e.g., antibodies or gelmatrix, e.g., polyacrylamide, which are used to purify it. The term“substantially purified” polypeptide of the present inventionconstitutes at least 50%, and often above 90%, of the purifiedpreparation as based on amino acid analysis.

The term “acid” for the purposes of the present invention, refers to anyorganic acid that is capable of hydrolyzing contaminating proteins whileallowing silk or other structural proteins to remain intact. Formic,acetic, propionic, butyric, and valeric acids are all examples oforganic acids, although other acids are also contemplated.

For the purposes of this invention, we define a “protein” as a polymerin which the monomers are amino acids and which are joined togetherthrough amide bonds and alternatively referred to as a polypeptide. Theterms “protein” and “polypeptide” are herein used interchangeably.Standard abbreviations for amino acids are used (e.g., P for proline).These abbreviations are included in Stryer, Biochemistry, Third Ed.,(1988), which is incorporated herein by reference for all purposes.

The term “silk polypeptide” refers to a protein that approximates themolecular and structural profile of native silk proteins and fibers.

The term “structural protein” or “structural polypeptide” refers to aclass of non-catalytic proteins that may serve as a biologicalstructural support. The proteins may serve as biological structuralsupports by themselves, in conjunction with other proteins, or as amatrix or support for other materials. Examples from this class include,but are not limited to, proteins such as spider silks ,that are used forspider web architecture; porin proteins, which form channels inbiological membranes; keratin, the major structural component of hair;collagen, the major extracellular protein in connective tissue. Thesilks, OmpF and recognin proteins described herein are examples ofstructural proteins.

The term “recovered” refers to the process by which protein is locallysequestered and captured.

The term “organic acid” refers to the class of acids, such as formic,acetic, propionic, butyric, and valeric acids, which are found in livingorganisms but not necessarily, derived from said living organism. Saidorganic acids can also be obtained from commercial vendors (e.g. SigmaChemical).

As used herein, the term “host cell” refers to any cell capable ofexpressing a functional gene and/or gene product introduced from anothercell or organism. This definition includes E. coli., as well as otherorganisms.

The term “insoluble fibers” refers to proteinaceous fibers that will notsolubilize in an aqueous solution.

The term “bacterial” refers to any of numerous groups of microscopic,one-celled organisms including, but not limited to the phylum Eubacteriaof the kingdom Procaryotae.

The term “concentrating” refers to any process that increases themolarity of proteinaceous solution.

The term “concentrated solution” refers to a proteinaceous solutionadjusted to a predetermined molarity higher than said pre-adjustedproteinaceous solution.

The term “spinneret” refers to a small orifice used for fiber formation.

DESCRIPTION OF THE INVENTION

A number of different embodiments, as exemplified in the examples, ofthe present invention are contemplated, including the scaling-up of themethod, automation of the method, or use of the method to purify otherstructural proteins.

One of skill in the art will recognize that the practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology,microbiology, and recombinant DNA, which are within the skill of theart. Such techniques are described in the literature. See, for example,Molecular Cloning, A Laboratory, Manual, 2nd Ed., by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J.Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.).

The proteins of the present invention can be made by direct synthesis(chemically or biologically) or by expression from cloned DNA. Thesource of the protein is not limited to recombinant means.Non-recombinant proteins may be purified or spun using the methodsdescribed herein. Indeed, Example 6, infra, describes the purificationof E. coli OmpF membrane protein, which is, in this case, a naturallyoccurring (i.e. non-recombinant protein) protein.

The means for expressing cloned DNA are generally known in the art.However, there are some considerations for design of expression vectorsthat are unusual for expressing DNA encoding the spider silk proteins ofthe present invention. For example, the proteins are highly repetitivein their structure. Accordingly, cloned DNA should be propagated andexpressed in host cell strains that will maintain repetitive sequencesin extrachromosomal elements (e.g. SURE™ cells, Stratagene). Also, dueto the high content of alanine, glycine, proline, and glutamine, itmight be advantageous to use a host cell which over expresses tRNA forthese amino acids.

The present invention contemplates the use of many different organicacids to manipulate recombinant and non-recombinant biological samplesunder conditions such that a polypeptide is substantially purified.While the use of E. coli cells with formic, propionic and valeric acidare contemplated, the present invention is not limited to theseparticular embodiments, but may also be practiced using other organicacids, such as acetic, and butyric, acids, all of which serve asexamples. The present invention may also be practiced using otherprokaryotic or eukaryotic cells (aside from, or along with, E. colicells), the media in which the protein-of-interest has been secreted,organs, tissue, bone and other components, all of which are examples ofbiological sample materials.

EXPERIMENTAL

The following examples serve to illustrate certain embodiments andaspects of the present invention and are not to be construed as limitingthe scope thereof.

EXAMPLE 1 Purification of Recombinant Silk Protein with Formic Acid andIon Exchange Chromatography

In this example, the gene product of pQE(sp1)₇ (SEQ ID NO: 1), as setout in FIG. 1, is expressed as recombinant silk protein pQE(SP1)₇ (SEQID NO: 2), as set out in FIG. 2, in E.coli as described elsewhere(Prince et al., 1995). The (sp1)₇ gene was cloned into the expressionvector pQE-9 (Qiagen) and transformed into the host cell lineSG13009pREP4 (Stratagene). Cultures were grown to an A₆₀₀=1.5-2.0 in4×YT medium (per liter: 32 g tryptone, 20 g yeast extract, 5 g NaCl)containing 400 ug/mL ampicillin. Protein expression was induced by theaddition of isopropyl-thiogalactopyranoside (IPTG) to a finalconcentration of 1 mM. After 1-4 hours the cells were harvested bycentrifugation and stored for purification. Lyophilized pellets werelysed in 23N formic acid (100 ml/g cell pellet), diluted to 4.6N acidwith distilled and deionized water and stirred 1 hour at roomtemperature. The cell lysate was clarified by centrifugation andconcentrated 20 fold by ultrafiltration. The solution was clarified bycentrifugation and the supernatant was dialyzed extensively into 8Murea, 10 mM NaH₂PO₄, 1 mM Tris, 20 mM NaCl, pH 8. Precipitated materialwas removed by centrifugation and the clarified supernatant was appliedto an affinity chromatography resin (nickel-NTA agarose) that had beenequilibrated with 8M urea, 10 mM NaH₂PO₄, 1 mM Tris, 20 mM NaCl, pH 8.The chromatography conditions were designed to bind the recombinant silkprotein, but let the remaining bacterial proteins pass through thecolumn. The column was washed with 8M urea, 10 mM NaH₂PO₄, 1 mM Tris, 20mM NaCl, pH 7. The pQE(SP1)₇ protein was eluted from the column 8M urea,10 mM NaH₂PO₄, 1 mM Tris, 20 mM NaCl, pH 3.The sample was 94% pure asdetermined by quantitative amino acid analysis. FIG. 12 illustrates acomparison of traditional purification techniques with the methodologyenclosed in this application. Cells lysed with formic acid yielded moresilk protein with a similar purity when compared to the 6M guanidinelysis with Ni-NTA affinity chromatography.

EXAMPLE 2 Purification of Recombinant Silk Protein with Propionic Acidand Ion Exchange Chromatography

In this example, the gene product of pQE[(SP1)₄/(SP2)₁]₄ (SEQ ID NO: 3),as set out in FIG. 3, is expressed as recombinant silk proteinpQE[(SP1)₄/(SP2)₁]₄ (SEQ ID NO: 4), as set out in FIG. 4, in E.coli(Prince et al., 1995). The [(SP1)₄/(SP2)₁]₄ gene was cloned into theexpression vector pQE-9 (Qiagen) and transformed into the host cellSG13009pREP4 (Stratagene). Cultures were grown to an A₆₀₀=1.5-2.0 in4×YT medium (per liter: 32 g tryptone, 20 g yeast extract, 5 g NaCl)containing 400 ug/mL ampicillin. Protein expression was induced by theaddition of IPTG to a final concentration of 1 mM. After 1-4 hours thecells were harvested by centrifugation and stored for purification.Lyophilized pellets were lysed in 13.3N propionic acid (2 ml/g cellpellet), diluted to 2.3N acid with distilled and deionized water andstirred 1 hour at room temperature. The cell lysate was clarified bycentrifugation and concentrated 20 fold by ultrafiltration. Many of theacid stable proteins became insoluble and were removed bycentnifugation. The clarified supernatant was dialyzed extensively into10 mM Tris, pH 9 containing 2M urea. The dialyzed solution was appliedto a strong anion exchange resin, QAE-Sephadex A50, that had beenequilibrated with 10 mM Tris, pH 9 containing 2M urea. Thechromatography conditions were designed such that the positively chargedsilk protein would not bind to the column, but the remaining proteinswith lower isoelectric points and net negative charge would bind to thecolumn. The column was washed with 10 mM Tris, pH 9 containing 2M ureato recover any remaining silk protein. The wash was pooled with theunbound silk containing fraction and processed. The sample was 97% pureas determined by quantitative amino acid analysis.

EXAMPLE 3 Purification of Recombinant Silk Protein with Propionic Acidand Ion Exchange Chromatography

In this example, the gene product of pET[(SP1)₄/(SP2)₁]₄ (SEQ ID NO: 5),as set out in FIG. 5, is expressed as recombinant silk proteinpET[(SP1)₄/(SP2)₁]₄ (SEQ ID NO: 6), as set out in FIG. 6, in E.coli(Prince et al., 1995). The [(SP1)₄/ (SP2)₁]₄ gene was cloned into theexpression vector pET24 (Novagen Inc.) and transformed into the hostcell BL21(DE3) pLysS. Cultures were grown to an A₆₀₀=19 in definedmedium (per liter: 13.3 g KH₂PO₄, 4 g (NH₄)₂HPO₄, 1.7 g Citric acid, 25g glucose, 1,2 g MgSO₄-7H₂O, 39 mg FeCl₃, 13 mg MnSO₄-H₂O, 10 mgZnSO₄-7H₂O, 3 mg H₃BO₃, 2.5 mg Na₂MoO₄-2 H₂O, 2.5 mg CoCl₂-6H₂O, 1.8 mgCu(CH₃COO)₂-H₂O, 6.7 mg EDTA, 4.5 mg thiamine-HCl) with kanamycin (30ug/mI) at 37° C., 16 liter/min air and 600 rpm. Expression was inducedfor 1 hr with 1 mM IPTG at which time the cells were harvested bycentrifugation and stored for purification. Lyophilized pellets werelysed in 13.3N propionic acid (2 ml/g cell pellet), diluted to 2.3N acidwith distilled and deionized water and stirred 1 hour at roomtemperature. The cell lysate was clarified by centrifugation andconcentrated 20 fold by ultrafiltration. Many of the acid stableproteins became insoluble and were removed by centrifugation. Theclarified supernatant was dialyzed extensively into 10 mM Tris, pH 9containing 2M urea. The dialyzed solution was applied to a strong anionexchange resin QAE-Sephadex A50 that had been equilibrated with 10 mMTris, pH 9 containing 2M urea. The chromatography conditions weredesigned such that the positively charged silk protein would not bind tothe column, but the remaining proteins with lower isoelectric points andnet negative charge would bind to the column. The column was washed with10 mM Tris, pH 9 containing 2M urea to recover any remaining silkprotein. The wash was pooled with the unbound silk containing fractionand processed. The sample was 75-85% pure as determined bycoomassie-blue staining of a polyacrylamide gel (see FIG. 13).

EXAMPLE 4 Purification of Recombinant Silk protein with Propionic AcidContaining Denaturant and Ion Exchange Chromatography

In this example, the gene product of pET[(SP1)₄/(SP2)₁]₄ (SEQ ID NO: 5),as set out in FIG. 5, is expressed as recombinant silk proteinpET[(SP1)₄/(SP2)₁]₄ (SEQ ID NO: 6), as set out in FIG. 6, in E.coli(Prince et al., 1995). Lyophilized pellets were lysed in 13.3N propionicacid (2 mL/g cell pellet), diluted to 2.3N acid with 6M guanidinehydrochloride (to a final concentration of 3M) and distilled anddeionized water and stirred for 1 hour at room temperature. The celllysate was clarified by centrifugation and concentrated 3 fold byultrafiltration. Precipitated material was removed by centrifugation andthe clarified supernatant was dialyzed extensively into 10 mM Tris, pH 9containing 2M urea. Many of the acid stable proteins became insolubleand were removed by centrifugation. The dialyzed supernatant was appliedto a strong anion exchange resin, QAE-Sephadex A50 that had beenequilibrated with 10 mM Tris, pH 9 containing 2M urea. Thechromatography conditions were designed such that the positively chargedsilk protein would not bind to the column, but the remaining proteinswith lower isoelectric points and net negative charge would bind to thecolumn. The column was washed with 10 mM Tris, pH 9 containing 2M ureato recover any remaining silk protein (see FIG. 14). The wash was pooledwith the unbound silk containing fraction and processed as describe inexample 9. This sample was approximately 80% pure based on coomassieblue staining.

EXAMPLE 5 Purification of Recombinant Silk Protein with Formic AcidContaining Denaturant and Affinity Chromatography

In this example, the gene product of pETNcDS (SEQ ID NO: 7), as set outin FIG. 7, is expressed as recombinant silk protein pETNcDS (SEQ ID NO:8), as set out in FIG. 8, in E.coli (Arcidiacono et al. 1998). The NcDSgene was cloned into the expression vector pET24 (Novagen Inc.) andtransformed into the host cell BL21(DE3) pLysS. Cultures were grown toan A₆₀₀=4 in 4×YT medium (per liter: 32 g tryptone, 20 g yeast extract,5 g NaCl) with kanamycin (30 ug/ml)at 37° C., 1 liter/min air and 800rpm. Expression was induced for 3 hr with 1 mM IPTG at which time thecells were harvested by centrifugation and stored for purification.Lyophilized pellets were lysed in 23N formic acid (5 ml/g cell pellet),diluted to 2.3N acid with 6M guanidine hydrochloride (to a finalconcentration of 3M) and distilled and deionized water and stirred 1hour at room temperature. The cell lysate was clarified bycentrifugation and concentrated 20 fold by ultrafiltration. The solutionwas clarified by centrifugation and the supernatant was dialyzedextensively into 8M urea, 10 mM NaH₂PO₄, 1 mM Tris, 20 mM NaCl, pH 8.Precipitated material was removed by centrifugation and the clarifiedsupernatant was applied to an affinity chromatography resin (nickel-NTAagarose) that had been equilibrated with 8M urea, 10 mM NaH₂PO₄, 1 mMTris, 20 mM NaCl, pH 8. The chromatography conditions were designed tobind the recombinant silk protein, but let the remaining bacterialproteins pass through the column. The column was washed with 8M urea, 10mM NaH₂PO₄, 1 mM Tris, 20 mM NaCl, pH 7. The NcDS protein was elutedfrom the column 8M urea, 10 mM NaH₂PO₄, 1 mM Tris, 20 mM NaCl, pH 3. Thepurified protein could then be processed for fiber spinning as inExample 8.

EXAMPLE 6 Valeric Acid Purification of E. coli OmpF Membrane Protein

In this example, a native E. coli ompF membrane protein (SEQ ID NO: 9),as presented in FIG. 9, was purified. Cells were grown and harvested asdescribed in Example 3. Because OmpF is a native E. coli protein, itsproduction was not induced by the addition of IPTG. Lyophilized pelletswere lysed in 9.2N valeric acid (2 mL/g of pellet), diluted to 2.3N acidwith distilled and deionized water and stirred for 1 hour at roomtemperature. The cell lysate was clarified by centrifugation and appliedto an SDS polyacrylamide gel for electrophoresis. FIG. 15 represents thepolyacrylamide gel depicting this purification of ompF, a native E. colistructural protein from a lyophilized E. coli cell pellet. The ompFprotein was than blotted onto a nitrocellulose membrane for N-terminalsequencing. The resulting 30 amino acids of N-terminal sequence led tothe identification of E. coli outer membrane protein, ompF. This simpleextraction procedure yielded a purity of approximately 85% based oncoomassie-blue staining.

EXAMPLE 7 Organic Acid Extraction of Recognin B1 Protein

In this example, the gene product of Recognin B1 (SEQ ID NO: 10), as setout in FIG. 10, was expressed as recombinant synthetic coiled proteinRecognin B1 (SEQ ID NO: 11), as set out in FIG. 11, in E.coli (McGrath,K. P. and Kaplan, D. L. Mat. Res. Symp. Proc. 292, 83-91). The RecogninB1 gene was cloned into the expression vector pQE-9 (Qiagen) andtransformed into the E. coli host cell, SG13009pREP4 (Qiagen). Cultureswere grown to an A₆₀₀ of 1 in 4×Yt medium (per liter: 32 g tryptone, 20g yeast extract, 5 g NaCl) with ampicillin (400 ug/mL) and kanamycin (50ug/mL). Expression was induced for two hours with 1 mM IPTG at whichtime the cells were harvested by centrifugation and stored forpurification. Individual lyophilized pellets were lysed separately in23N formic acid, 17.5N acetic acid, 13.4N propionic acid, 10.9N butyricacid or 9.2N valeric acid (2 mL/g of pellet), diluted to 2.3N acid withdistilled and deionized water and stirred for 1 hour at roomtemperature. The cell lysates were clarified by centrifugation andanalyzed by SDS-PAGE. FIG. 16 presents the polyacrylamide gel ofRecognin B1, a recombinant coiled coil structural protein. A cell pelletwas lysed in either gel loading buffer, formic acid or valeric acid.Relative amounts of the cell pellet loaded onto the gel were 85, 400,900 ug for the loading buffer, formic and valeric acid lysates,respectively. Acetic, propionic or butyric acids were unable to extractthis protein. The results indicated that formic and valeric acids wereable to extract a significant quantity of Recognin B1 from E. colipellets. The extracted protein did not appear to be degraded uponexposure to these organic acids. Of the two acids, valeric acid was ableto extract Recognin B1 in a relatively pure form.

EXAMPLE 8 Processing and Fiber Spinning of Recombinant Silk Protein

Recombinant pETNcDS protein was purified as in Example 5, concentrated100-fold by ultrafiltration and dialyzed into 10 mM NaH₂PO₄, 1 mM Tris,20 mM NaCl, pH5 containing 1 M urea. The dialyzed sample was clarifiedby centrifugation and concentrated by ultrafiltration to a 25% (w/w)solution for fiber spinning. A Harvard Apparatus Infusion/WithdrawalPump (Harvard Instruments, Natick MA) was used with a specializedmicrospinner (cavity volume 0.5 ml), and a 6 cm (0.005 I.D.) piece oftubing which was used as a spinneret. The silk solution was forcedthrough the spinneret at a rate of 5-10 ul/min into a coagulation bathconsisting of 90% methanol. Water insoluble fibers, 10-60 um indiameter, were produced and prepared for light microscopy (see FIG. 17).

EXAMPLE 9 Processing and Fiber Spinning the pET[(Sp1)₄/(Sp2)₁]₄Recombinant Silk Protein

pET[(SP1)₄/(SP2)₁]₄ (SEQ ID NO: 6) was purified as described in Example4. The sample wasclarified by centrifugation and concentrated byultrafiltration to 9.3% (w/w) solution for fiber spinning. A HarvardApparatus Infusion/Withdrawal Pump (Harvard Instruments, Natick MA) wasused with a specialized microspinner (cavity volume 0.5 ml) and a 6 cm(0.005 I.D.) piece of tubing which was used as a spinneret. The silksolution was forced through the spinneret at a rate of 2-5 ul/min into acoagulation bath consisting of 90% methanol. Fibers were produced fromthe solution. Fibers from the 9.3% solution were removed from thecoagulation bath: said fibers were water insoluble and were subsequentlyprepared for light microscopy.

EXAMPLE 10

Processing and Fiber Spinning the pQE[(Sp1)₄/(Sp2)₁]₄ Recombinant SilkProtein

The pQE[(Sp1)₄/(Sp2)₁]₄ protein was purified by lysis in formicacid/guanidine hydrochloride as in Example 5 and dialyzed into 10 mMNaH₂PO₄, 1 mM Tris, 20 mM NaCl, pH 5 containing 160 mM urea. Thedialyzed sample was clarified by centrifugation and concentrated byultrafiltration to 6.5% and 12.5% (w/w) solution for fiber spinning. AHarvard Apparatus Infusion/Withdrawal Pump (Harvard Instruments, NatickMA) was used with a specialized microspinner (cavity volume 0.5 ml) anda 6 cm (0.005′ I.D.) piece of tubing was used as a spinneret. The silksolution was forced through the spinneret at a rate of 5-10 ul/min intoa coagulation bath consisting of 90% methanol. Fibers were produced fromeach solution. Only fibers from the 12.5% solution could be removed fromthe coagulation bath; they were water insoluble and prepared for lightmicroscopy (see FIG. 18).

From the above description and examples, it should be clear that thepresent invention provides improved methods for purifying structuralproteins and spinning spider silk proteins. Accordingly, this inventionis not limited to the particular embodiments disclosed, but is intendedto cover all modifications that are within the spirit and scope of theinvention as defined by the appended claims.

11 1 876 DNA Nephila clavipes 1 atgagaggat cgcatcacca tcaccatcacggatccatgg ctagcggtag aggcgggctg 60 ggtggccagg gtgcaggtgc ggctgcggctgccgcggcag cggccgcagg cggtgccggc 120 caaggtggct atggcggcct gggttctcaggggactagcg gtagaggcgg gctgggtggc 180 cagggtgcag gtgcggctgc ggctgccgcggcagcggccg caggcggtgc cggccaaggt 240 ggctatggcg gcctgggttc tcaggggactagcggtagag gcgggctggg tggccagggt 300 gcaggtgcgg ctgcggctgc cgcggcagcggccgcaggcg gtgccggcca aggtggctat 360 ggcggcctgg gttctcaggg gactagcggtagaggcgggc tgggtggcca gggtgcaggt 420 gcggctgcgg ctgccgcggc agcggccgcaggcggtgccg gccaaggtgg ctatggcggc 480 ctgggttctc aggggactag cggtagaggcgggctgggtg gccagggtgc aggtgcggct 540 gcggctgccg cggcagcggc cgcaggcggtgccggccaag gtggctatgg cggcctgggt 600 tctcagggga ctagcggtag aggcgggctgggtggccagg gtgcaggtgc ggctgcggct 660 gccgcggcag cggccgcagg cggtgccggccaaggyggct atggcggcct gggttctcag 720 gggactagcg gtagaggcgg gctgggtggccagggtgcag gtgcggctgc ggctgccgcg 780 gcagcggccg caggcggtgc cggccaaggtggctatggcg gcctgggttc tcaggggact 840 agtgggatcc gtcgacctgc agccaagcttaattag 876 2 291 PRT Nephila clavipes 2 Met Arg Gly Ser His His His HisHis His Gly Ser Met Ala Ser Gly 1 5 10 15 Arg Gly Gly Leu Gly Gly GlnGly Ala Gly Ala Ala Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Gly Gly AlaGly Gln Gly Gly Tyr Gly Gly Leu Gly 35 40 45 Ser Gln Gly Thr Ser Gly ArgGly Gly Leu Gly Gly Gln Gly Ala Gly 50 55 60 Ala Ala Ala Ala Ala Ala AlaAla Ala Ala Gly Gly Ala Gly Gln Gly 65 70 75 80 Gly Tyr Gly Gly Leu GlySer Gln Gly Thr Ser Gly Arg Gly Gly Leu 85 90 95 Gly Gly Gln Gly Ala GlyAla Ala Ala Ala Ala Ala Ala Ala Ala Ala 100 105 110 Gly Gly Ala Gly GlnGly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Thr 115 120 125 Ser Gly Arg GlyGly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala 130 135 140 Ala Ala AlaAla Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly 145 150 155 160 LeuGly Ser Gln Gly Thr Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly 165 170 175Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly 180 185190 Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Thr Ser Gly Arg Gly 195200 205 Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala210 215 220 Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly SerGln 225 230 235 240 Gly Thr Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly AlaGly Ala Ala 245 250 255 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala GlyGln Gly Gly Tyr 260 265 270 Gly Gly Leu Gly Ser Gln Gly Thr Ser Gly IleArg Arg Pro Ala Ala 275 280 285 Lys Leu Asn 290 3 2046 DNA Nephilaclavipes 3 atgagaggat cgcatcacca tcaccatcac ggatccatgg ctagcggtagaggcgggctg 60 ggtggccagg gtgcaggtgc ggctgcggct gccgcggcag cggccgcaggcggtgccggc 120 caaggtggct atggcggcct gggttctcag gggactagcg gtagaggcgggctgggtggc 180 cagggtgcag gtgcggctgc ggctgccgcg gcagcggccg caggcggtgccggccaaggt 240 ggctatggcg gcctgggttc tcaggggact agcggtagag gcgggctgggtggccagggt 300 gcaggtgcgg ctgcggctgc cgcggcagcg gccgcaggcg gtgccggccaaggtggctat 360 ggcggcctgg gttctcaggg gactagcggt agaggcgggc tgggtggccagggtgcaggt 420 gcggctgcgg ctgccgcggc agcggccgca ggcggtgccg gccaaggtggctatggcggc 480 ctgggttctc aggggactag cggtccgggc ggttatggtc cgggtcaacaaactagcggt 540 agaggcgggc tgggtggcca gggtgcaggt gcggctgcgg ctgccgcggcagcggccgca 600 ggcggtgccg gccaaggtgg ctatggcggc ctgggttctc aggggactagcggtagaggc 660 gggctgggtg gccagggtgc aggtgcggct gcggctgccg cggcagcggccgcaggcggt 720 gccggccaag gtggctatgg cggcctgggt tctcagggga ctagcggtagaggcgggctg 780 ggtggccagg gtgcaggtgc ggctgcggct gccgcggcag cggccgcaggcggtgccggc 840 caaggtggct atggcggcct gggttctcag gggactagcg gtagaggcgggctgggtggc 900 cagggtgcag gtgcggctgc ggctgccgcg gcagcggccg caggcggtgccggccaaggt 960 ggctatggcg gcctgggttc tcaggggact agcggtccgg gcggttatggtccgggtcaa 1020 caaactagcg gtagaggcgg gctgggtggc cagggtgcag gtgcggctgcggctgccgcg 1080 gcagcggccg caggcggtgc cggccaaggt ggctatggcg gcctgggttctcaggggact 1140 agcggtagag gcgggctggg tggccagggt gcaggtgcgg ctgcggctgccgcggcagcg 1200 gccgcaggcg gtgccggcca aggtggctat ggcggcctgg gttctcaggggactagcggt 1260 agaggcgggc tgggtggcca gggtgcaggt gcggctgcgg ctgccgcggcagcggccgca 1320 ggcggtgccg gccaaggtgg ctatggcggc ctgggttctc aggggactagcggtagaggc 1380 gggctgggtg gccagggtgc aggtgcggct gcggctgccg cggcagcggccgcaggcggt 1440 gccggccaag gtggctatgg cggcctgggt tctcagggga ctagcggtccgggcggttat 1500 ggtccgggtc aacaaactag cggtagaggc gggctgggtg gccagggtgcaggtgcggct 1560 gcggctgccg cggcagcggc cgcaggcggt gccggccaag gtggctatggcggcctgggt 1620 tctcagggga ctagcggtag aggcgggctg ggtggccagg gtgcaggtgcggctgcggct 1680 gccgcggcag cggccgcagg cggtgccggc caaggtggct atggcggcctgggttctcag 1740 gggactagcg gtagaggcgg gctgggtggc cagggtgcag gtgcggctgcggctgccgcg 1800 gcagcggccg caggcggtgc cggccaaggt ggctatggcg gcctgggttctcaggggact 1860 agcggtagag gcgggctggg tggccagggt gcaggtgcgg ctgcggctgccgcggcagcg 1920 gccgcaggcg gtgccggcca aggtggctat ggcggcctgg gttctcaggggactagcggt 1980 ccgggcggtt atggtccggg tcaacaaact agtgggatcc gtcgacctgcagccaagctt 2040 aattag 2046 4 681 PRT Nephila clavipes 4 Met Arg Gly SerHis His His His His His Gly Ser Met Ala Ser Gly 1 5 10 15 Arg Gly GlyLeu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala 20 25 30 Ala Ala AlaAla Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly 35 40 45 Ser Gln GlyThr Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly 50 55 60 Ala Ala AlaAla Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly 65 70 75 80 Gly TyrGly Gly Leu Gly Ser Gln Gly Thr Ser Gly Arg Gly Gly Leu 85 90 95 Gly GlyGln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 100 105 110 GlyGly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Thr 115 120 125Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala 130 135140 Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly 145150 155 160 Leu Gly Ser Gln Gly Thr Ser Gly Pro Gly Gly Tyr Gly Pro GlyGln 165 170 175 Gln Thr Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala GlyAla Ala 180 185 190 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly GlnGly Gly Tyr 195 200 205 Gly Gly Leu Gly Ser Gln Gly Thr Ser Gly Arg GlyGly Leu Gly Gly 210 215 220 Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala AlaAla Ala Ala Gly Gly 225 230 235 240 Ala Gly Gln Gly Gly Tyr Gly Gly LeuGly Ser Gln Gly Thr Ser Gly 245 250 255 Arg Gly Gly Leu Gly Gly Gln GlyAla Gly Ala Ala Ala Ala Ala Ala 260 265 270 Ala Ala Ala Ala Gly Gly AlaGly Gln Gly Gly Tyr Gly Gly Leu Gly 275 280 285 Ser Gln Gly Thr Ser GlyArg Gly Gly Leu Gly Gly Gln Gly Ala Gly 290 295 300 Ala Ala Ala Ala AlaAla Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly 305 310 315 320 Gly Tyr GlyGly Leu Gly Ser Gln Gly Thr Ser Gly Pro Gly Gly Tyr 325 330 335 Gly ProGly Gln Gln Thr Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly 340 345 350 AlaGly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly 355 360 365Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Thr Ser Gly Arg Gly 370 375380 Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala 385390 395 400 Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly SerGln 405 410 415 Gly Thr Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala GlyAla Ala 420 425 430 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly GlnGly Gly Tyr 435 440 445 Gly Gly Leu Gly Ser Gln Gly Thr Ser Gly Arg GlyGly Leu Gly Gly 450 455 460 Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala AlaAla Ala Ala Gly Gly 465 470 475 480 Ala Gly Gln Gly Gly Tyr Gly Gly LeuGly Ser Gln Gly Thr Ser Gly 485 490 495 Pro Gly Gly Tyr Gly Pro Gly GlnGln Thr Ser Gly Arg Gly Gly Leu 500 505 510 Gly Gly Gln Gly Ala Gly AlaAla Ala Ala Ala Ala Ala Ala Ala Ala 515 520 525 Gly Gly Ala Gly Gln GlyGly Tyr Gly Gly Leu Gly Ser Gln Gly Thr 530 535 540 Ser Gly Arg Gly GlyLeu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala 545 550 555 560 Ala Ala AlaAla Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly 565 570 575 Leu GlySer Gln Gly Thr Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly 580 585 590 AlaGly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly 595 600 605Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Thr Ser Gly Arg Gly 610 615620 Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala 625630 635 640 Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly SerGln 645 650 655 Gly Thr Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln ThrSer Gly 660 665 670 Ile Arg Arg Pro Ala Ala Lys Leu Asn 675 680 5 2076DNA Nephila clavipes 5 atggctagca tgactggtgg acagcaaatg ggtcgcggatccatggctag cggtagaggc 60 gggctgggtg gccagggtgc aggtgcggct gcggctgccgcggcagcggc cgcaggcggt 120 gccggccaag gtggctatgg cggcctgggt tctcaggggactagcggtag aggcgggctg 180 ggtggccagg gtgcaggtgc ggctgcggct gccgcggcagcggccgcagg cggtgccggc 240 caaggtggct atggcggcct gggttctcag gggactagcggtagaggcgg gctgggtggc 300 cagggtgcag gtgcggctgc ggctgccgcg gcagcggccgcaggcggtgc cggccaaggt 360 ggctatggcg gcctgggttc tcaggggact agcggtagaggcgggctggg tggccagggt 420 gcaggtgcgg ctgcggctgc cgcggcagcg gccgcaggcggtgccggcca aggtggctat 480 ggcggcctgg gttctcaggg gactagcggt ccgggcggttatggtccggg tcaacaaact 540 agcggtagag gcgggctggg tggccagggt gcaggtgcggctgcggctgc cgcggcagcg 600 gccgcaggcg gtgccggcca aggtggctat ggcggcctgggttctcaggg gactagcggt 660 agaggcgggc tgggtggcca gggtgcaggt gcggctgcggctgccgcggc agcggccgca 720 ggcggtgccg gccaaggtgg ctatggcggc ctgggttctcaggggactag cggtagaggc 780 gggctgggtg gccagggtgc aggtgcggct gcggctgccgcggcagcggc cgcaggcggt 840 gccggccaag gtggctatgg cggcctgggt tctcaggggactagcggtag aggcgggctg 900 ggtggccagg gtgcaggtgc ggctgcggct gccgcggcagcggccgcagg cggtgccggc 960 caaggtggct atggcggcct gggttctcag gggactagcggtccgggcgg ttatggtccg 1020 ggtcaacaaa ctagcggtag aggcgggctg ggtggccagggtgcaggtgc ggctgcggct 1080 gccgcggcag cggccgcagg cggtgccggc caaggtggctatggcggcct gggttctcag 1140 gggactagcg gtagaggcgg gctgggtggc cagggtgcaggtgcggctgc ggctgccgcg 1200 gcagcggccg caggcggtgc cggccaaggt ggctatggcggcctgggttc tcaggggact 1260 agcggtagag gcgggctggg tggccagggt gcaggtgcggctgcggctgc cgcggcagcg 1320 gccgcaggcg gtgccggcca aggtggctat ggcggcctgggttctcaggg gactagcggt 1380 agaggcgggc tgggtggcca gggtgcaggt gcggctgcggctgccgcggc agcggccgca 1440 ggcggtgccg gccaaggtgg ctatggcggc ctgggttctcaggggactag cggtccgggc 1500 ggttatggtc cgggtcaaca aactagcggt agaggcgggctgggtggcca gggtgcaggt 1560 gcggctgcgg ctgccgcggc agcggccgca ggcggtgccggccaaggtgg ctatggcggc 1620 ctgggttctc aggggactag cggtagaggc gggctgggtggccagggtgc aggtgcggct 1680 gcggctgccg cggcagcggc cgcaggcggt gccggccaaggtggctatgg cggcctgggt 1740 tctcagggga ctagcggtag aggcgggctg ggtggccagggtgcaggtgc ggctgcggct 1800 gccgcggcag cggccgcagg cggtgccggc caaggtggctatggcggcct gggttctcag 1860 gggactagcg gtagaggcgg gctgggtggc cagggtgcaggtgcggctgc ggctgccgcg 1920 gcagcggccg caggcggtgc cggccaaggt ggctatggcggcctgggttc tcaggggact 1980 agcggtccgg gcggttatgg tccgggtcaa caaactagtgggatccgaat tcgagctccg 2040 tcgacaagct tcgagcacca ccaccaccac cactga 20766 691 PRT Nephila clavipes 6 Met Ala Ser Met Thr Gly Gly Gln Gln Met GlyArg Gly Ser Met Ala 1 5 10 15 Ser Gly Arg Gly Gly Leu Gly Gly Gln GlyAla Gly Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Gly Gly Ala GlyGln Gly Gly Tyr Gly Gly 35 40 45 Leu Gly Ser Gln Gly Thr Ser Gly Arg GlyGly Leu Gly Gly Gln Gly 50 55 60 Ala Gly Ala Ala Ala Ala Ala Ala Ala AlaAla Ala Gly Gly Ala Gly 65 70 75 80 Gln Gly Gly Tyr Gly Gly Leu Gly SerGln Gly Thr Ser Gly Arg Gly 85 90 95 Gly Leu Gly Gly Gln Gly Ala Gly AlaAla Ala Ala Ala Ala Ala Ala 100 105 110 Ala Ala Gly Gly Ala Gly Gln GlyGly Tyr Gly Gly Leu Gly Ser Gln 115 120 125 Gly Thr Ser Gly Arg Gly GlyLeu Gly Gly Gln Gly Ala Gly Ala Ala 130 135 140 Ala Ala Ala Ala Ala AlaAla Ala Gly Gly Ala Gly Gln Gly Gly Tyr 145 150 155 160 Gly Gly Leu GlySer Gln Gly Thr Ser Gly Pro Gly Gly Tyr Gly Pro 165 170 175 Gly Gln GlnThr Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly 180 185 190 Ala AlaAla Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly 195 200 205 GlyTyr Gly Gly Leu Gly Ser Gln Gly Thr Ser Gly Arg Gly Gly Leu 210 215 220Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 225 230235 240 Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Thr245 250 255 Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala AlaAla 260 265 270 Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly TyrGly Gly 275 280 285 Leu Gly Ser Gln Gly Thr Ser Gly Arg Gly Gly Leu GlyGly Gln Gly 290 295 300 Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala AlaGly Gly Ala Gly 305 310 315 320 Gln Gly Gly Tyr Gly Gly Leu Gly Ser GlnGly Thr Ser Gly Pro Gly 325 330 335 Gly Tyr Gly Pro Gly Gln Gln Thr SerGly Arg Gly Gly Leu Gly Gly 340 345 350 Gln Gly Ala Gly Ala Ala Ala AlaAla Ala Ala Ala Ala Ala Gly Gly 355 360 365 Ala Gly Gln Gly Gly Tyr GlyGly Leu Gly Ser Gln Gly Thr Ser Gly 370 375 380 Arg Gly Gly Leu Gly GlyGln Gly Ala Gly Ala Ala Ala Ala Ala Ala 385 390 395 400 Ala Ala Ala AlaGly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly 405 410 415 Ser Gln GlyThr Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly 420 425 430 Ala AlaAla Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly 435 440 445 GlyTyr Gly Gly Leu Gly Ser Gln Gly Thr Ser Gly Arg Gly Gly Leu 450 455 460Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 465 470475 480 Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Thr485 490 495 Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Thr Ser Gly ArgGly 500 505 510 Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala AlaAla Ala 515 520 525 Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly LeuGly Ser Gln 530 535 540 Gly Thr Ser Gly Arg Gly Gly Leu Gly Gly Gln GlyAla Gly Ala Ala 545 550 555 560 Ala Ala Ala Ala Ala Ala Ala Ala Gly GlyAla Gly Gln Gly Gly Tyr 565 570 575 Gly Gly Leu Gly Ser Gln Gly Thr SerGly Arg Gly Gly Leu Gly Gly 580 585 590 Gln Gly Ala Gly Ala Ala Ala AlaAla Ala Ala Ala Ala Ala Gly Gly 595 600 605 Ala Gly Gln Gly Gly Tyr GlyGly Leu Gly Ser Gln Gly Thr Ser Gly 610 615 620 Arg Gly Gly Leu Gly GlyGln Gly Ala Gly Ala Ala Ala Ala Ala Ala 625 630 635 640 Ala Ala Ala AlaGly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly 645 650 655 Ser Gln GlyThr Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gln Gln Thr 660 665 670 Ser GlyIle Arg Ile Arg Ala Pro Ser Thr Ser Phe Glu His His His 675 680 685 HisHis His 690 7 1588 DNA Nephila clavipes 7 atggctagca tgactggtggacagcaaatg ggtcggatcc gaattcgtgg atatggaggt 60 cttggtggac aaggtgccggacaaggagct ggtgcagccg ccgcagcagc agctggtggt 120 gccggacaag gaggatatggaggtcttgga agccaaggtg ctggacgagg tggacaaggt 180 gcaggcgcag ccgcagccgcagctggaggt gctggtcaag gaggatacgg aggtcttgga 240 agccaaggtg ctggacgaggaggattaggt ggacaaggtg caggtgcagc agcagcagct 300 ggaggtgtcg gacaaggaggactaggtgga caaggtgctg gacaaggagc tggagcagct 360 gctgcagcag ctggtggtgccggacaagga ggatatggag gtctcggaag ccaaggtgca 420 ggacgaggtg gatcaggtggacaaggggca ggtgcagcag cagcagcagc tggaggtgcc 480 ggacaaggag gatatggaggtcttggaagc caaggtgcag gacgaggtgg attaggtgga 540 cagggtgcag gtgcagcagcagcagcagca gccggaggtg ctggacaagg aggatacggt 600 ggtcttggtg gacaaggtgccggacaaggt ggctatggag gacttggaag ccaaggtgct 660 ggacgaggag gattaggtggacaaggtgca ggtgcagcag cagcagctgg aggtgccgga 720 caaggaggac taggtggacaaggagctgga gcagccgctg cagcagctgg tggtgccgga 780 caaggaggat atggaggtcttggaagccaa ggtgctggac gaggtggaca aggtgcaggc 840 gcagccgcag cagcagccggaggtgctgga caaggaggat acggtggaca aggtgccgga 900 caaggaggct atggaggacttggaagccaa ggtgctggac gaggaggatt aggtggacaa 960 ggtgcaggtg cagcagcagcagcagcagca gctggaggtg ccggacaagg aggattaggt 1020 ggacaaggtg caggtgcagcagcagcagca gctggaggtg ctggacaagg aggattaggt 1080 ggacaaggtg ctggacaaggagctggagca gccgctgcag cagccgctgc agcagctggt 1140 ggtgttagac aaggaggatatggaggtctt ggaagccaag gtgctggacg aggtggacaa 1200 ggtgcaggcg cagccgcagcagcagccgga ggtgctggac aaggaggata tggtggtctt 1260 ggtggacaag gtgttggacgaggtggatta ggtggacaag gtgcaggcgc agcggcagct 1320 gttggtgctg gacaaggaggatatggtggt gttggttctg gggcgtctgc tgcctctgca 1380 gctgcatccc gtttgtcttctcctcaagct agttcaagag tttcatcagc tgtttccaac 1440 ttggttgcaa gtggtcctactaattctgcg gccttgtcaa gtacaatcag taatgtggtt 1500 tcacaaatag gcgccagcaatcctggtctt tctggatgtg atgtcctcat tcaagctctt 1560 ctcgagcacc accaccaccaccactgaa 1588 8 528 PRT Nephila clavipes 8 Met Ala Ser Met Thr Gly GlyGln Gln Met Gly Arg Ile Arg Ile Arg 1 5 10 15 Gly Tyr Gly Gly Leu GlyGly Gln Gly Ala Gly Gln Gly Ala Gly Ala 20 25 30 Ala Ala Ala Ala Ala AlaGly Gly Ala Gly Gln Gly Gly Tyr Gly Gly 35 40 45 Leu Gly Ser Gln Gly AlaGly Arg Gly Gly Gln Gly Ala Gly Ala Ala 50 55 60 Ala Ala Ala Ala Gly GlyAla Gly Gln Gly Gly Tyr Gly Gly Leu Gly 65 70 75 80 Ser Gln Gly Ala GlyArg Gly Gly Leu Gly Gly Gln Gly Ala Gly Ala 85 90 95 Ala Ala Ala Ala GlyGly Val Gly Gln Gly Gly Leu Gly Gly Gln Gly 100 105 110 Ala Gly Gln GlyAla Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly 115 120 125 Gln Gly GlyTyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg Gly Gly 130 135 140 Ser GlyGly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala 145 150 155 160Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg Gly 165 170175 Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Gly 180185 190 Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala Gly195 200 205 Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg GlyGly 210 215 220 Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Gly GlyAla Gly 225 230 235 240 Gln Gly Gly Leu Gly Gly Gln Gly Ala Gly Ala AlaAla Ala Ala Ala 245 250 255 Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly LeuGly Ser Gln Gly Ala 260 265 270 Gly Arg Gly Gly Gln Gly Ala Gly Ala AlaAla Ala Ala Ala Gly Gly 275 280 285 Ala Gly Gln Gly Gly Tyr Gly Gly GlnGly Ala Gly Gln Gly Gly Tyr 290 295 300 Gly Gly Leu Gly Ser Gln Gly AlaGly Arg Gly Gly Leu Gly Gly Gln 305 310 315 320 Gly Ala Gly Ala Ala AlaAla Ala Ala Ala Ala Gly Gly Ala Gly Gln 325 330 335 Gly Gly Leu Gly GlyGln Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly 340 345 350 Gly Ala Gly GlnGly Gly Leu Gly Gly Gln Gly Ala Gly Gln Gly Ala 355 360 365 Gly Ala AlaAla Ala Ala Ala Ala Ala Ala Ala Gly Gly Val Arg Gln 370 375 380 Gly GlyTyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Arg Gly Gly Gln 385 390 395 400Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly 405 410415 Thr Gly Gly Leu Gly Gly Gln Gly Val Gly Ala Gly Gly Leu Gly Gly 420425 430 Gln Gly Ala Gly Ala Ala Ala Ala Val Gly Ala Gly Gln Gly Gly Tyr435 440 445 Gly Gly Val Gly Ser Gly Ala Ser Ala Ala Ser Ala Ala Ala SerArg 450 455 460 Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala ValSer Asn 465 470 475 480 Leu Val Ala Ser Gly Pro Thr Asn Ser Ala Ala LeuSer Ser Thr Ile 485 490 495 Ser Asn Val Val Ser Gln Ile Gly Ala Ser AsnPro Gly Leu Ser Gly 500 505 510 Cys Asp Val Leu Ile Gln Ala Leu Leu GlyHis His His His His His 515 520 525 9 341 PRT Nephila clavipes 9 Ala GluIle Tyr Asn Lys Asp Gly Asn Lys Val Asp Leu Tyr Gly Lys 1 5 10 15 AlaVal Gly Leu His Tyr Phe Ser Lys Gly Asn Gly Glu Asn Ser Tyr 20 25 30 GlyGly Asn Gly Asp Met Thr Tyr Ala Arg Leu Gly Phe Lys Gly Glu 35 40 45 ThrGln Ile Asn Ser Asp Leu Thr Gly Tyr Gly Gln Trp Glu Tyr Asn 50 55 60 PheGln Gly Asn Asn Ser Glu Gly Ala Asp Ala Gln Thr Gly Asn Lys 65 70 75 80Thr Arg Leu Ala Phe Ala Gly Leu Lys Tyr Ala Asp Val Gly Ser Phe 85 90 95Asp Tyr Gly Arg Asn Tyr Gly Val Val Tyr Asp Ala Leu Gly Tyr Thr 100 105110 Asp Met Leu Pro Glu Phe Gly Gly Asp Thr Ala Tyr Ser Asp Asp Phe 115120 125 Phe Val Gly Arg Val Gly Gly Val Ala Thr Tyr Arg Asn Ser Asn Phe130 135 140 Phe Gly Leu Val Asp Gly Leu Asn Phe Ala Val Gln Tyr Leu GlyLys 145 150 155 160 Asn Glu Arg Asp Thr Ala Arg Arg Ser Asn Gly Asp GlyVal Gly Gly 165 170 175 Ser Ile Ser Tyr Glu Tyr Glu Gly Phe Gly Ile ValGly Ala Tyr Gly 180 185 190 Ala Ala Asp Arg Thr Asn Leu Gln Glu Ala GlnPro Leu Gly Asn Gly 195 200 205 Lys Lys Ala Glu Gln Trp Ala Thr Gly LeuLys Tyr Asp Ala Asn Asn 210 215 220 Ile Tyr Leu Ala Ala Asn Tyr Gly GluThr Arg Asn Ala Thr Pro Ile 225 230 235 240 Thr Asn Lys Phe Thr Asn ThrSer Gly Phe Ala Asn Lys Thr Gln Asp 245 250 255 Val Leu Leu Val Ala GlnTyr Gln Phe Asp Phe Gly Leu Arg Pro Ser 260 265 270 Ile Ala Tyr Thr LysSer Lys Ala Lys Asp Val Glu Gly Ile Gly Asp 275 280 285 Val Asp Leu ValAsn Tyr Phe Glu Val Gly Ala Thr Tyr Tyr Phe Asn 290 295 300 Lys Asn MetSer Thr Tyr Val Asp Tyr Ile Ile Asn Gln Ile Asp Ser 305 310 315 320 AspAsn Lys Leu Gly Val Gly Ser Asp Asp Thr Val Ala Val Gly Ile 325 330 335Val Tyr Gln Phe Ala 340 10 225 DNA Nephila clavipes 10 atgagaggatcgcatcacca tcaccatcac ggatccatgg ctagcggtga cctgaaaaac 60 aaagtggcccagctgaaaag gaaagttaga tctctgaaag ataaagcggc tgaactgaaa 120 caagaagtctcgagactgga aaatgaaatc gaagacctga aagccaaaat tggtgacctg 180 aataacactagtgggatccg tcgacctgca gccaagctta attag 225 11 74 PRT Nephila clavipes 11Met Arg Gly Ser His His His His His His Gly Ser Met Ala Ser Gly 1 5 1015 Asp Leu Lys Asn Lys Val Ala Gln Leu Lys Arg Lys Val Arg Ser Leu 20 2530 Lys Asp Lys Ala Ala Glu Leu Lys Gln Glu Val Ser Arg Leu Glu Asn 35 4045 Glu Ile Glu Asp Leu Lys Ala Lys Ile Gly Asp Leu Asn Asn Thr Ser 50 5560 Gly Ile Arg Arg Pro Ala Ala Lys Leu Asn 65 70

We claim:
 1. A method, comprising: (a) providing: i) a biological samplecomprising one or more structural polypeptides; and ii) an acidconsisting essentially of an organic acid; (b) treating said sample withsaid acid under conditions such that said one or more structuralpolypeptides are purified and recovered in a solution, said treatingcomprising mixing said sample with said acid, incubating said mixedsample and acid, and clarifying said mixed sample and acid to yield therecovered solution comprising said one or more purified polypeptides. 2.The method of claim 1, wherein said polypeptide is selected from SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, and SEQID NO:
 11. 3. The method of claim 1, wherein said biological samplecomprises recombinant polypeptides.
 4. The method of claim 1, whereinsaid biological sample comprises non-recombinant polypeptides.
 5. Themethod of claim 1, wherein said acid is an organic acid.
 6. The methodof claim 5, wherein said organic acid is selected from formic, acetic,propionic, butyric, and valeric acids.
 7. The method of claim 1, furthercomprising the steps of purifying the solution, concentrating thepurified solution, and forcing the concentrated solution through aspinneret under conditions wherein insoluble fibers of polypeptides areproduced.
 8. A method, comprising: (a) providing: i) host cellsexpressing one or more recombinant structural polypeptides, and ii) asolution consisting essentially of an organic acid; and (b) treatingsaid host cells with said solution to create a mixture of soluble andinsoluble material, whereby said treating comprises mixing said cellswith said acid, incubating said mixed cells and acid, and clarifyingsaid mixed cells and acid to yield a recovered solution, said recoveredsolution comprising one or more purified polypeptides.
 9. The method ofclaim 8, wherein said one or more polypeptides is selected from SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, and SEQID NO:
 11. 10. The method of claim 8, wherein said organic acid isselected from formic acid, acetic acid, propionic acid, butyric acid,and valeric acid.
 11. The method of claim 8, further comprisingpurifying the solution, concentrating the purified solution, and forcingthe concentrated solution through a spinneret under conditions whereininsoluble fibers of polypeptides are produced.
 12. A method, comprising:(a) providing: i) bacterial cells expressing one or more recombinantstructural polypeptides, and ii) a solution consisting essentially of anorganic acid selected from formic acid, acetic acid, propionic acid,butyric acid, and valeric acid; and (b) treating said bacterial cellswith said solution to create a mixture of soluble and insolublematerial, whereby said treating comprises mixing said cells with saidacid, incubating said mixed cells and acid, and clarifying said mixedcells and acid to yield a recovered solution, said recovered solutioncomprising one or more purified polypeptides.
 13. The method of claim12, wherein said one or more polypeptides is selected from SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO:
 11. 14. Themethod of claim 12, further comprising the steps of purifying thesolution, concentrating said recovered one or more recombinantpolypeptides to create a concentrated purified solution, and forcingsaid concentrated solution through a spinneret under conditions whereininsoluble fibers of polypeptides are produced.